Application Of HCV Replicon To Screen Anti-hepatitis C Chinese Traditional Medicine Formula

Hepatitis C is a global epidemic disease. The World Health Organization(WHO) has estimated that approximately3%of the world population havebeen infected with the virus. In China, approximately40million individuals(3.2%of the population) have been diagnosed with hepatitis C, most of thatdo not develop obvious clinical symptoms, even at the beginning of theinfection. Hepatitis C infection, which is prone to chronicity, has a long andaggressive disease history, and usually results in progressive hepatic injuryand fibrosis, culminating in cirrhosis and even hepatocellular carcinoma.Therefore, the aim for treatment of hepatitis C is the clearance of the virus.Nowadays, the combination of interferon and ribavirin are therecommended optimal treatment, while the virological response only occurs inapproximately50–60%of patients. However, the adverse effects of thisregimen is frequent and large numbers of patients are not suitable candidatesfor treatment for a variety of reasons. Currently protease inhibitor has beenemerging as new complementary and alternative medicine for anti-HCV.Using protease inhibitors as monotherapy is prone to develop resistencewithin a few days or weeks. Herein, there is an urgent need for thedevelopment of novel therapy strategies. Some Chinese herb has been used inthe therapy of viral hepatitis, however, the study focus on the method toscreen the suitble formula for anti-HCV is still limited. Moreover, nude miceinoculation tests as classical recommended method has many drawbacks, suchas cost-intensive, labour-consuming, and rarely applied.In the present study, HCV replicon cell lines sh7(H7-HCV-L7), whichcan express both neomycin phosphotransferase and Renilla luciferase, areselected as a model to screen for anti-HCV medicine using the plasmapharmacology and experimental methods. Our study was divided into two parts. In the first part, we explored the effect of animal plasma on culturedcells to determine animal species and the appropriate concentration of theplasma. In the second part, we screened16Chinese herbal medicines alongwith prescribed formula and verified the anti-HCV effects of them.Pa rt I: The effect of animal plasma on the cultured cellsObjective: To prepare adaptive cell lines mH7-HCV-L7from inoculatednude mice and select appropriate animal plasma and determine plasmaconcentrations.Methods:1Preparation of nude mice adaptive cell lines mH7-HCV-L7and validation.H7-HCV-L7cells were transplanted in nude mice subcutaneously. The micewere raised for another two months after the tumor was developed. And thetumor was resected under sterile conditions and isolated as mH7-HCV-L7primary culture cells. CCK-8method was employed to measure thecytotoxicity of different concentration (10%,20%,30%,40%) mice plasmaagainst H7-HCV-L7and mH7-HCV-L7at different hours (24h,48h,72h).2The H7-HCV-L7cells were treated with the plasma of the SPF KM mice,Wistar rats, New Zealand rabbit, at different concentrations (10%,20%,30%,40%)and for different time(24h,48h,72h). Then we analyzed the cytotoxicityof the different plasma on cultured cells to determine the appropiate animalspecie and optimal plasma concentration as assayed by CCK-8method.Results:1Comparison of the toxicity of mouse plasma against H7-HCV-L7andmH7-HCV-L7cells1.1With the increased concentration and the prolonged duration of mouseplasma, the optical density (OD) of H7-HCV-L7and mH7-HCV-L7wasdecreased, suggesting the toxicity of mouse plasma increased;1.2The OD value of mH7-HCV-L7cells was slightly higher than that ofH7-HCV-L7, at24h and48h after incubation with10%mouse plasma. However, there was no significant difference observed at other concentrationsand time points,1.3Cell survival rate was75.30%,57.29%and21.12%at24h,48h,72hrespectively using10%mouse plasma, suggesting this concentration stillremained comparatively higher toxicity to mH7-HCV-L7cells.2Cytotoxicity comparison of plasma from mouse, rat and rabbit to H7-HCV-L7cells.2.1Comparision of the OD value of mH7-HCV-L7cells treating withdifferent plasma at the same time point and with the same concentration.Results showed that the mouse plasma had relatively lower cytotoxicity, ratplasma was moderate, and rabbit plasma was the most toxic to cells.2.2There was no significant difference among the OD value of the differentgroups at48h and24h. While the OD obtained from30%and40%rabbitplasma groups and the DMEM control group was statistically different at72hand24h (P<0.05).2.3After incubation48h, there was no statistically significant difference of theOD value of rabbit serum treated cells (concentration of10%,20%and30%,respectively) and that of DMEM cultured cells as control (P>0.05). Thegrowth of H7-HCV-L7cells treated with40%rabbit serum was apparentlyinhibited.Conclusion:1The mH7-HCV-L7cells adapted in nude mice can hardly withstand lowerconcentrations of mouse plasma;2The rabbit plasma is most suitable for the growth of H7-HCV-L7cells, andthe optimal incubation duration was48h. In order to detect the effect ofChinese herbs on cell growth, we chose20%as final rabbit plasmaconcentration to extend the subquent screening tests of anti-HCV TCM. Part II: Application of HCV replicon to screen anti-hepatitis C ChineseTraditional Medicine formulaObjective: To screen16candidate traditional Chinese medicine as wellas the prescribed formula for its high viral eradication.Methods:1Within HCV replicon cell lines,16candidate Chinese medicine werescreened (Saxiftaga melanocentra Franch, Stem of Sargentgloryvine, Root ofCairo Morningglory, Plumbago zeylanica L, Maytenus hookeri Loes, Fruit ofCape Jasmine, Salvia yunnanensis C.H.Wright, Root of Lightyellow Sophorn,Rheum tanguticum Maxim.ex Balf, Radix Sophorae Tonkinensis,Phyllanthusnirud Linn, Lonicera hypoglauca Miq, Radix Gentianae, BaicalSkullcap Root, Cinnamomi Cortex, Fructus Ligustri Lucidi). The traditionalChinese medicine plasma pharmacology methold was adopted.1.1Preparation of drug–containing rabbit plasma and rabbit plasma as blankcontrol.1.2To get the standard curve of H7-HCV-L7with48-well plate by the CCK-8procedure.1.3To inoculate cells in48-well plates, and culture for24hours followed bymedium change with DMEM plus20%drug-containing rabbit plasma or20%blank rabbit plasma. Incubation for48hours;1.4Alignment of OD values, cell survival rate and the cell numbers in eachwell by the CCK-8procedure.1.5To measure the fluorescence density in each well, and to calculatefluorescence of single cell in each group with Renilla luciferase light kit andGlomax20/20luminescence detecter;1.6To calculate anti-HCV efficency of these traditional Chinese medicines,and to rank them by inhibition rate.2To select the prescriptions (composed of effective traditional Chinesemedicines) with higher inhibitory rate and verify the effect.2.1To select prescriptions based on cytotoxicity, inhibition rate and traditionalChinese medicine property:Prescription One: Stem of Sargentgloryvine12g, Root of CairoMorningglory8g, Maytenus hookeri Loes45g, Baical Skullcap Root6g Prescription Two: Fructus Ligustri Lucidi10g, Fructus SchisandraeChinensis5g, Radix Astragali20g, Radix Codonopsis10gPrescription Three: Stem of Sargentgloryvine12g, Root of CairoMorningglory8g, Maytenus hookeri Loes45g, Baical Skullcap Root6g,Fructus Ligustri Lucidi10g, Fructus Schisandrae Chinensis5g, RadixAstragali20g, Radix Codonopsis10g2.2To measure the anti-HCV inhibitory rate of TCD composing prescriptionsand make comparison, according to the procedures in section2.1.3To further validate the higher inhibitory rate of TCD and prescriptions.3.1To extract RNA from the H7-HCV-L7cells treated by the three Chinesemedicine prescriptions and the four TCD agents in Prescription One (Stem ofSargentgloryvine, Root of Cairo Morningglory, Maytenus hookeri Loes,Baical Skullcap Root) respectively for48hours.3.2To measure the HCV RNA copy number (copies/ml) among all the groupswith the quantitative real-time PCR.3.3To quantify the RNA concentration (μg/ml) with protein nucleic acidanalysis instrument, calculate the average HCV RNA copy number containedin each microgram of RNA, calculate inhibition rate, and rank.Results:1The inhibition rate calculated by the single cell fluorescence values:20%rabbit plasma containing Stem of Sargentgloryvine, Root of CairoMorningglory, Maytenus hookeri Loes and Baical Skullcap Root showedhigher inhibitory rates of51.73%,43.46%,32.62%,23.68%respectively, andshowed no significant cytotoxicity. The Prescription Three showed highestHCV inhibition rate, the Prescription Two showed lowest rate, and theinhibition rate of the Prescription One was slightly lower than that of thePrescription Three.2Based on the inhibition rate of HCV RNA copy number in each microgramof total RNA, single herb demonstrated the highest inhibition rate. Theranking was the same: Stem of Sargentgloryvine, Root of Cairo Morningglory,Maytenus hookeri Loes, Baical Skullcap Root. However the composite descriptions showed lower inhibition rate. And the rank from the highest tothe lowest was Prescription Three, Prescription One, and Prescription Two.Conclusion: By the plasma pharmacology and experimental researchwith the HCV replicon, anti-HCV TCD were screened and the prescriptionswere preliminarily composed. Both the Prescription Three and thePrescription One were effective for anti-HCV. However, the Prescription Onewas unsuitable for long term use. Therefore, the Prescription Three was theopimal choice of anti-HCV in our study.